Reprogramming of glucose and glutamine metabolism is a key event in malignant transformation. However, the functional drivers and molecular pathways underlying these adaptations are only now starting to emerge. Our long-term goal is to understand mechanisms of metabolic reprogramming in tumor cells so that these processes can be targeted by molecular cancer therapeutics. The overall objective of the current application is to apply systems approaches to elucidate the functional role of the glutaminolytic phenotype elicited by overexpression of the Myc oncoprotein. Myc is an important regulator of both aerobic glycolysis as well as glutaminolysis in tumor cells, the latter of which is characterized by avid consumption of the non-essential amino acid glutamine. Our central hypothesis is that the glutaminolytic phenotype induced by Myc overexpression is important for combating oxidative stress and fueling lipid synthesis in rapidly dividing cells. The rationale for the proposed research is that, once we have identified the functional significance of Myc- induced metabolic reprogramming, these processes can be targeted to undermine the advantages they confer on tumor cells. We plan to test our central hypothesis by pursuing the following specific aims: (1) to determine the role of glutaminolysis in detoxification of reactive oxygen species and (2) to determine the role of glutaminolysis in promoting lipid synthesis. The proposed research is innovative because it applies systems approaches to quantitatively elucidate the global behavior of integrated metabolic networks, rather than individual reactions or nodes in isolation. We will apply metabolic flux analysis (MFA) to quantify metabolic phenotypes in P493-6 Burkitt's lymphoma cells as well as mouse embryonic fibroblasts that are dependent on Myc for their malignant transformation. These cells contain Myc constructs that can be modulated to achieve tuned levels of overexpression. The cells will be subjected to different treatments intended to elucidate the role of glycolysis and glutaminolysis in both cellular redox balancing and anaplerotic processes. In particular, we will simultaneously quantify flux through all three major routes of cytosolic NADPH production, which are important for maintenance of redox homeostasis in proliferating cells. Furthermore, we will investigate the regulation of pathways that serve to convert glucose and glutamine into lipid intermediates required to support cell growth. As a result of this work, we expect to contribute a deeper understanding of how Myc regulates cellular metabolism and how these adaptations provide a growth advantage to tumor cells. This is significant because it will stimulate the search for novel drug targets that can inhibit the progression of Myc-dependent tumors. This work will also provide an avenue toward personalized medicine by identifying the unique metabolic signatures of Myc overexpression. PUBLIC HEALTH RELEVANCE: The proposed research will have an important positive impact in the search for novel drug targets for cancer therapy. In addition, it will provide fundamental insights into the nature of metabolic reprogramming in cancer cells and how these events contribute to malignancy. Because direct inhibitors of Myc are not currently available to clinicians, reversal of Myc-induced metabolic phenotypes may provide a surrogate strategy to suppress the growth of Myc-dependent tumors.